Characterization of a calmodulin-binding transporter from the plasma membrane of barley aleurone.
We have used Arabidopsis calmodulin (CaM) covalently coupled to horseradish peroxidase to screen a barley aleurone cDNA expression library for CaM binding proteins. The deduced amino acid sequence of one cDNA obtained by this screen was shown to be a unique protein of 702 amino acids with CaM and cyclic nucleotide binding domains at the carboxyl terminus and high similarity to olfactory and K+ channels. This cDNA was designated HvCBT1 (Hordeum vulgare CaM binding transporter). Hydropathy plots of HvCBT1 showed the presence of six putative transmembrane domains, but sequence alignment indicated a pore domain that was unlike the consensus domains in K+ and olfactory channels. Expression of a subclone of amino acids 482-702 in Escherichia coli generated a peptide that bound CaM. When a fusion protein of HvCBT1 and green fluorescent protein was expressed in barley aleurone protoplasts, fluorescence accumulated in the plasma membrane. Expression of HvCBT1 in the K+ transport deficient Saccharomyces cerevisiae mutant CY162 showed no rescue of the mutant phenotype. However, growth of CY162 expressing HvCBT1 with its pore mutated to GYGD, the consensus sequence of K+ channels, was compromised. We interpret these data as indicating that HvCBT1 acts to interfere with ion transport.
Schuurink
R C
RC
Department of Plant and Microbial Biology, University of California, Berkeley, CA 94720-3102, USA.
Shartzer
S F
SF
Fath
A
A
Jones
R L
RL
D000595:Amino Acid Sequence; D001692:Biological Transport; D002148:Calmodulin-Binding Proteins / Q000235:genetics / Q000302:isolation & purification*; D002462:Cell Membrane / Q000737:chemistry; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D001467:Hordeum / Q000737:chemistry* / Q000235:genetics; D007473:Ion Channels / Q000235:genetics; D008565:Membrane Proteins / Q000235:genetics / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000235:genetics / Q000302:isolation & purification*
C110848:CBT1 protein, Hordeum vulgare; D002148:Calmodulin-Binding Proteins; D018076:DNA, Complementary; D007473:Ion Channels; D008565:Membrane Proteins; D010940:Plant Proteins